Tissue transglutaminases (TGases) are GTP-binding/GTPases with an enzymatic transamidation activity that catalyzes the covalent linkage between glutamine residues and primary amino groups, resulting in the formation of new protein-protein and protein-polyamine complexes. TGases have been implicated in a number of biologically important processes including cellular differentiation and apoptosis. TGase expression levels, GTP-binding and transamidation activities in various cell lines were shown to increase in response to treatment with retinoic acid (RA). We recently discovered that two proteins crucial for cellular differentiation and survival, Rb and eukaryotic initiation factor 5A (eIF5A), are targets of TGase. RA-dependent protection against apoptosis in the HL-60 cell line has been linked to TGase mediated transamidation of Rb, protecting it from caspase cleavage. A major goal of this work will be to characterize the interactions of TGase with these new targets, the main focus being Rb. First we plan to establish if TGase mediated protection of Rb is dependent upon TGase mediated transamidation, or if it is due to direct binding between the two proteins. Mutants of Rb that lack the TGase transamidation or binding site will be used to investigate the role of the TGase/Rb interaction in PA or growth factor-stimulated protection from apoptosis, and cellular differentiation. We also will analyze the cellular localization of TGase, and in particular, determine whether TGase localization changes in response to RA treatment or is influenced by its GTP-binding/GTP hydrolytic cycle. Overall, the results of these studies will provide biochemical detail as to how TGase interaction with its substrates affects cell growth and differentiation.